Abstract Ion transfer across biological membranes is central to nerve excitation, muscle cell contraction, signal transduction, and hormone secretion. Ion channels play a vital role by providing a passageway ? the ion conduction pore ? within membranes to allow specific ions to traverse down their electrochemical gradient. The immense physiological importance of ion channels is reflected in the fact that their dysfunction underlies a variety of disabling human diseases including seizures, deafness, ataxia, long QT syndrome, and cardiac arrhythmias. There is a long history of physiological work and a large body of functional and structural data on tetrameric cation channels that are localized to the plasma membrane, including the K+, Ca2+, Na+, TRP and cyclic nucleotide-gated (CNG) channels. However, relatively little is known about organellar cation channels, partly because of the difficulty in directly measuring their activities in organellar membranes. Currently, there is an emerging research interest in the recently defined two-pore channels (TPCs) due to their importance in endo/lysosome physiology. In human and animals, TPC channels regulate the ionic homeostasis and pH within lysosomes, set the lysosomal membrane potential and excitability, and may also regulate the lysosomal Ca2+ release. Therefore, TPC channel functions directly or indirectly affect lysosome-mediated processes such as cellular degradation as well as catabolite export and trafficking, and defects of these processes can result in lysosomal storage diseases. Thus, understanding the molecular basis of TPC channel functions will provide basic, fundamental knowledge about many TPC-related lysosomal activities and diseases. My laboratory has a longstanding interest in studying the structural basis of ion selectivity and gating, the two fundamental properties of tetrameric cation channels. Our studies over the last funding period have been focused specifically on deciphering the ion selectivity properties in K+ and CNG channels using the prokaryotic non-selective NaK channel from Bacillus cereus as a model system. In this proposal, we plan to expand our research to eukaryotic organellar TPC channels. More specifically, our proposed research will center on a plant vacuolar TPC1 channel from Arabidopsis thaliana, AtTPC1, which is a non-selective cation channel regulated by both membrane potential and Ca2+, whose structure was determined recently in my lab. A combined approach of protein crystallography and electrophysiology will be employed in the proposed studies.